1,721,051 research outputs found
Synthesis of activated 3′-amino-3′-deoxy-2-thio-thymidine, a superior substrate for the nonenzymatic copying of nucleic acid templates
No full textWe present a scalable synthesis of 3'-amino-3'-deoxy-2-thio-thymidine-5'-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the rate of template copying by 5-fold compared with the 3'-amino-3'-deoxy-T monomer, while the 3'-amino monomers exhibit a 2- to 3'-fold enhancement compared with their ribonucleotide counterparts.1120sciescopu
In Vitro Selection of pH‐Activated DNA Nanostructures
No full textWe report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH-active nanostructures were designed using known pH-active motifs, such as the i-motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH-selectivity, releasing up to 714-fold more Mipomersen at pH5.2 compared to pH7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH-sensitive motifs, suggesting that they may operate via novel structure-switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH-active surfaces.1141sciescopu
Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline
No full textA novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 mu M. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.112419sciescopu
In vitro selection of shape-changing DNA nanostructures capable of binding-induced cargo release
No full textMany biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate 'cargo strand' containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 mu M, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications.1175sciescopu
In vitro selection of structure-switching, self-reporting aptamers
No full textWe describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.116361sciescopu
Thiolated uridine substrates and templates improve the rate and fidelity of ribozyme-catalyzed RNA copying
No full textRibozyme-catalyzed RNA polymerization is inefficient and error prone. Here we demonstrate that two alternative bases, 2-thio-uridine (s(2)U) and 2-thio-ribo-thymidine (s(2)T), improve the rate and fidelity of ribozyme catalyzed nucleotide addition as NTP substrates and as template bases. We also demonstrate the functionality of s(2)U and s(2)T-containing ribozymes.1111sciescopu
Thousand-fold volumetric concentration of live cells with a recirculating acoustofluidic device
No full textThe ability to concentrate cells from dilute samples into smaller volumes is an essential process step for most biological assays. Volumetric concentration is typically achieved via centrifugation, but this technique is not well suited for handling small number of cells, especially outside of the laboratory setting. In this work, we describe a novel device that combines acoustofluidics with a recirculating architecture to achieve >1000-fold enrichment of cells in a label-free manner, at high volumetric throughput (>500 mu L, min(-1)) and with high recovery (>98.7%). We demonstrate that our device can be used with a wide variety of different cell types and show that this concentration strategy does not affect cell viability. Importantly, our device could be readily adopted to serve as a "sample preparation" module that can be integrated with other microfluidic devices to allow analysis of dilute cellular samples in large volumes.111711sciescopu
Detection of proteins in serum by micromagnetic aptamer PCR (MAP) Technology
No full text116971sciescopu
Probing the limits of aptamer affinity with a microfluidic SELEX platform
No full textNucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K-d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K-d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K-d.115453sciescopu
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